PubMed Health⌕ Search

Biomedical subjects

Gregory C Amberg

Publications and source records attributed to Gregory C Amberg.

13 recordsLinked to original sources
PubMed2006

Calcium sparklets regulate local and global calcium in murine arterial smooth muscle.

In arterial smooth muscle, protein kinase Calpha (PKCalpha) coerces discrete clusters of L-type Ca2+ channels to operate in a high open probability mode, resulting in subcellular domains of nearly continual Ca2+ influx called 'persistent Ca2+ sparklets'. Our previous work suggested that steady-state Ca2+ entry into arterial myocytes, and thus global [Ca2+]i, is regulated by Ca2+ influx through clusters of L-type Ca2+ channels operating in this persistently active mode in addition to openings of solitary channels functioning in a low-activity mode. Here, we provide the first direct evidence supporting this 'Ca2+ sparklet' model of Ca2+ influx at a physiological membrane potential and external Ca2+ concentration. In support of this model, we found that persistent Ca2+ sparklets produced local and global elevations in [Ca2+]i. Membrane depolarization increased Ca2+ influx via low-activity and high-activity persistent Ca2+ sparklets. Our data indicate that Ca2+ entering arterial smooth muscle through persistent Ca2+ sparklets accounts for approximately 50% of the total dihydropyridine-sensitive (i.e. L-type Ca2+ channel) Ca2+ influx at a physiologically relevant membrane potential (-40 mV) and external Ca2+ concentration (2 mm). Consistent with this, inhibition of basal PKCalpha-dependent persistent Ca2+ sparklets decreased [Ca2+]i by about 50% in isolated arterial myocytes and intact pressurized arteries. Taken together, these data support the conclusion that in arterial smooth muscle steady-state Ca2+ entry and global [Ca2+]i are regulated by low-activity and PKCalpha-dependent high-activity persistent Ca(2+) sparklets.

Animals↗
PubMed2006

Activation of NFATc3 down-regulates the beta1 subunit of large conductance, calcium-activated K+ channels in arterial smooth muscle and contributes to hypertension.

Large conductance, Ca2+-activated K+ (BK) channels modulate the excitability and contractile state of arterial smooth muscle. Recently, we demonstrated that during hypertension, expression of the accessory beta1 subunit was decreased relative to the pore-forming alpha subunit of the BK channel. Reduced beta1 subunit expression resulted in BK channels with impaired function due to lowered sensitivity to Ca2+. Here, we tested the hypothesis that activation of the calcineurin/NFATc3 signaling pathway down-regulates beta1 expression during angiotensin II-induced hypertension. Consistent with this hypothesis, we found that in vivo administration of angiotensin II-activated calcineurin/NFATc3 signaling in arterial smooth muscle. During angiotensin II infusion, arterial smooth muscle BK channel function was decreased in wild type (WT) but not in NFATc3 null (NFATc3-/-) mice. Accordingly, beta1 expression was decreased in WT but not in NFATc3-/- arteries. Angiotensin II-induced down-regulation of the beta1 subunit required Ca2+ influx via L-type Ca2+ channels. However, in the absence of angiotensin II, moderate elevation of [Ca2+]i alone was not sufficient to activate NFAT transcriptional activity and, thus, decrease beta1 subunit expression. Importantly, angiotensin II infusion increased systemic blood pressure to a lower extent in NFATc3-/- than in WT mice, indicating that this transcription factor is required for the development of severe hypertension during chronic angiotensin II signaling activation. We conclude that activation of calcineurin and NFATc3 during sustained angiotensin II signaling down-regulates the expression of the beta1 subunit of the BK channel, which in turn contributes to arterial dysfunction and the development of hypertension.

Angiotensin II↗
PubMed2006

Mechanisms underlying heterogeneous Ca2+ sparklet activity in arterial smooth muscle.

In arterial smooth muscle, single or small clusters of Ca(2+) channels operate in a high probability mode, creating sites of nearly continual Ca(2+) influx (called "persistent Ca(2+) sparklet" sites). Persistent Ca(2+) sparklet activity varies regionally within any given cell. At present, the molecular identity of the Ca(2+) channels underlying Ca(2+) sparklets and the mechanisms that give rise to their spatial heterogeneity remain unclear. Here, we used total internal reflection fluorescence (TIRF) microscopy to directly investigate these issues. We found that tsA-201 cells expressing L-type Cavalpha1.2 channels recapitulated the general features of Ca(2+) sparklets in cerebral arterial myocytes, including amplitude of quantal event, voltage dependencies, gating modalities, and pharmacology. Furthermore, PKCalpha activity was required for basal persistent Ca(2+) sparklet activity in arterial myocytes and tsA-201 cells. In arterial myocytes, inhibition of protein phosphatase 2A (PP2A) and 2B (PP2B; calcineurin) increased Ca(2+) influx by evoking new persistent Ca(2+) sparklet sites and by increasing the activity of previously active sites. The actions of PP2A and PP2B inhibition on Ca(2+) sparklets required PKC activity, indicating that these phosphatases opposed PKC-mediated phosphorylation. Together, these data unequivocally demonstrate that persistent Ca(2+) sparklet activity is a fundamental property of L-type Ca(2+) channels when associated with PKC. Our findings support a novel model in which the gating modality of L-type Ca(2+) channels vary regionally within a cell depending on the relative activities of nearby PKCalpha, PP2A, and PP2B.

Animals↗
PubMed2006

Kv2 channels oppose myogenic constriction of rat cerebral arteries.

By hyperpolarizing arterial smooth muscle, voltage-gated, Ca2+-independent K+ (Kv) channels decrease calcium influx and thus oppose constriction. However, the molecular nature of the Kv channels function in arterial smooth muscle remains controversial. Recent investigations have emphasized a predominant role of Kv1 channels in regulating arterial tone. In this study, we tested the hypothesis Kv2 channels may also significantly regulate tone of rat cerebral arteries. We found that Kv2.1 transcript and protein are present in cerebral arterial smooth muscle. In addition, our analysis indicates that a substantial component (approximately 50%) of the voltage dependencies and kinetics of Kv currents in voltage-clamped cerebral arterial myocytes is consistent with Kv2 channels. Accordingly, we found that stromatoxin, a specific inhibitor of Kv2 channels, significantly decreased Kv currents in these cells. Furthermore, stromatoxin enhanced myogenic constriction of pressurized arterial segments. We also found that during angiotensin II-induced hypertension, Kv2 channel function was reduced in isolated myocytes and in intact arteries. This suggests that impaired Kv2 channel activity may contribute to arterial dysfunction during hypertension. On the basis of these novel observations, we propose a new model of Kv channel function in arterial smooth muscle in which Kv2 channels (in combination with Kv1 channels) contribute to membrane hyperpolarization and thus oppose constriction.

Animals↗
PubMed2005

Constitutively active L-type Ca2+ channels.

Ca(2+) influx through L-type Ca(2+) channels (LTCCs) influences numerous physiological processes ranging from contraction in muscle and memory in neurons to gene expression in many cell types. However, the spatiotemporal organization of functional LTCCs has been nearly impossible to investigate because of methodological limitations. Here, we examined LTCC function with high temporal and spatial resolution using evanescent field fluorescence microscopy. Surprisingly, we found that LTCCs operated in functionally organized clusters, not necessarily as individual proteins. Furthermore, LTCC function in these clusters does not appear to be controlled by simple stochastic gating but instead by a PKC-dependent switch mechanism. This work suggests that resting intracellular free calcium concentration in arterial myocytes is predominantly controlled by this process in combination with rare voltage-dependent openings of individual LTCCs. We propose that Ca(2+) influx via persistent LTCCs may be an important mechanism regulating steady-state local and global Ca(2+) signals.

Animals↗
PubMed2004

Regulation of Kv4.3 currents by Ca2+/calmodulin-dependent protein kinase II.

The voltage-dependent K+ channel 4.3 (Kv4.3) is one of the major molecular correlates encoding a class of rapidly inactivating K+ currents, including the transient outward current in the heart (Ito) and A currents (IA) in neuronal and smooth muscle preparations. Recent studies have shown that Ito in human atrial myocytes and IA in murine colonic myocytes are modulated by Ca2+/calmodulin-dependent protein kinase II (CaMKII); however, the molecular target of CaMKII in these studies has not been elucidated. We performed experiments to investigate whether CaMKII could regulate Kv4.3 currents directly. Inclusion of the autothiophosphorylated form of CaMKII in the patch pipette (10 nM) prolonged Kv4.3 currents such that the time required to reach 50% inactivation from peak more than doubled, with positive shifts in voltage dependence of both activation and inactivation. In contrast, the rate of recovery from inactivation was accelerated under these conditions. CaMKII-inhibitory peptide or KN-93 produced effects opposite to that above; thus the rate of inactivation was increased, and recovery from inactivation decreased. A number of mutagenesis experiments were conducted on the three candidate CaMKII consensus sequence sites on the channel. Mutations at S550A, located at the COOH-terminal region of the channel, resulted in currents that inactivated more rapidly but recovered from inactivation at a slower rate than that of wild-type controls. In addition, these currents were unaffected by dialysis with either autothiophosphorylated CaMKII or the specific inhibitory peptide of CaMKII, suggesting that CaMKII slows the inactivation and accelerates the rate of recovery from inactivation of Kv4.3 currents by a direct effect at S550A, located at the COOH-terminal region of the channel.

Animals↗
PubMed2004

NFATc3 regulates Kv2.1 expression in arterial smooth muscle.

Voltage-gated K+ (Kv) channels control the excitability of arterial smooth muscle. However, the molecular mechanisms regulating Kv channel function in smooth muscle remain unclear. We examined the hypothesis that the vasoactive peptide angiotensin II (Ang II) regulates arterial smooth muscle Kv channel function via calcineurin-dependent activation of the transcription factor NFAT. We found that sustained administration of Ang II decreased Kv currents (IKv) by reducing the expression of Kv2.1 K+ channel subunits. This effect of Ang II was independent of pressure but required Ca2+ influx through L-type Ca2+ channels. Consistent with our hypothesis, we found that calcineurin and NFATc3 are obligatory components of the signaling cascade mediating reduced IKv by Ang II. We conclude that sustained Ang II exposure increases smooth muscle Ca2+, which leads to activation of calcineurin and NFATc3, culminating in decreased Kv2.1 expression and reduced IKv function. These results support the novel concept that NFATc3 controls the excitability of arterial smooth muscle by regulating Kv2.1 expression.

Adenoviridae↗
PubMed2003

Downregulation of the BK channel beta1 subunit in genetic hypertension.

The molecular mechanisms underlying increased arterial tone during hypertension are unclear. In vascular smooth muscle, localized Ca2+ release events through ryanodine-sensitive channels located in the sarcoplasmic reticulum (Ca2+ sparks) activate large-conductance, Ca2+-sensitive K+ (BK) channels. Ca2+ sparks and BK channels provide a negative feedback mechanism that hyperpolarizes smooth muscle and thereby opposes vasoconstriction. In this study, we examined Ca2+ sparks and BK channel function in Wistar-Kyoto (WKY) rats with borderline hypertension and in spontaneously hypertensive rats (SHR), a widely used genetic model of severe hypertension. We found that the amplitude of spontaneous BK currents in WKY and SHR cells were smaller than in normotensive cells even though Ca2+ sparks were of similar magnitude. BK channels in WKY and SHR cells were less sensitive to physiological changes in intracellular Ca2+ than normotensive cells. Our data indicate that decreased expression of the BK channel beta1 subunit underlies the lower Ca2+ sensitivity of BK channels in SHR and WKY myocytes. We conclude that the lower expression of the beta1 subunit during genetic borderline and severe hypertension reduced BK channel activity by decreasing the sensitivity of these channels to physiological changes in Ca2+. These results support the view that changes in the molecular composition of BK channels may be a fundamental event contributing to the development of vascular dysfunction during hypertension.

Animals↗
PubMed2003

A-type potassium currents in smooth muscle.

A-type currents are voltage-gated, calcium-independent potassium (Kv) currents that undergo rapid activation and inactivation. Commonly associated with neuronal and cardiac cell-types, A-type currents have also been identified and characterized in vascular, genitourinary, and gastrointestinal smooth muscle cells. This review examines the molecular identity, biophysical properties, pharmacology, regulation, and physiological function of smooth muscle A-type currents. In general, this review is intended to facilitate the comparison of A-type currents present in different smooth muscles by providing a comprehensive report of the literature to date. This approach should also aid in the identification of areas of research requiring further attention.

Animals↗
PubMed2003

Modulation of the molecular composition of large conductance, Ca(2+) activated K(+) channels in vascular smooth muscle during hypertension.

Hypertension is a clinical syndrome characterized by increased vascular tone. However, the molecular mechanisms underlying vascular dysfunction during acquired hypertension remain unresolved. Localized intracellular Ca2+ release events through ryanodine receptors (Ca2+ sparks) in the sarcoplasmic reticulum are tightly coupled to the activation of large-conductance, Ca2+-activated K+ (BK) channels to provide a hyperpolarizing influence that opposes vasoconstriction. In this study we tested the hypothesis that a reduction in Ca2+ spark-BK channel coupling underlies vascular smooth muscle dysfunction during acquired hypertension. We found that in hypertension, expression of the beta1 subunit was decreased relative to the pore-forming alpha subunit of the BK channel. Consequently, the BK channels were functionally uncoupled from Ca2+ sparks. Consistent with this, the contribution of BK channels to vascular tone was reduced during hypertension. We conclude that downregulation of the beta1 subunit of the BK channel contributes to vascular dysfunction in hypertension. These results support the novel concept that changes in BK channel subunit composition regulate arterial smooth muscle function.

Animals↗
PubMed2002

Contribution of Kv4 channels toward the A-type potassium current in murine colonic myocytes.

A rapidly inactivating K(+) current (A-type current; I(A)) present in murine colonic myocytes is important in maintaining physiological patterns of slow wave electrical activity. The kinetic profile of colonic I(A) resembles that of Kv4-derived currents. We examined the contribution of Kv4 alpha-subunits to I(A) in the murine colon using pharmacological, molecular and immunohistochemical approaches. The divalent cation Cd(2+) decreased peak I(A) and shifted the voltage dependence of activation and inactivation to more depolarized potentials. Similar results were observed with La(3+). Colonic I(A) was sensitive to low micromolar concentrations of flecainide (IC(50) = 11 microM). Quantitative PCR indicated that in colonic and jejunal tissue, Kv4.3 transcripts demonstrate greater relative abundance than transcripts encoding Kv4.1 or Kv4.2. Antibodies revealed greater Kv4.3-like immunoreactivity than Kv4.2-like immunoreactivity in colonic myocytes. Kv4-like immunoreactivity was less evident in jejunal myocytes. To address this finding, we examined the expression of K(+) channel-interacting proteins (KChIPs), which act as positive modulators of Kv4-mediated currents. Qualitative PCR identified transcripts encoding the four known members of the KChIP family in isolated colonic and jejunal myocytes. However, the relative abundance of KChIP transcript was 2.6-fold greater in colon tissue than in jejunum, as assessed by quantitative PCR, with KChIP1 showing predominance. This observation is in accordance with the amplitude of the A-type current present in these two tissues, where colonic myocytes possess densities twice that of jejunal myocytes. From this we conclude that Kv4.3, in association with KChIP1, is the major molecular determinant of I(A) in murine colonic myocytes.

Animals↗
PubMed2002

Characterization of the A-type potassium current in murine gastric antrum.

A-type currents are rapidly inactivating potassium currents that operate at subthreshold potentials. A-type currents have not been reported to occur in the phasic muscles of the stomach. We used conventional voltage-clamp techniques to identify and characterize A-type currents in myocytes isolated from the murine antrum. A-type currents were robust in these cells, with peak current densities averaging 30 pA pF(-1) at 0 mV. These currents underwent rapid inactivation with a time constant of 83 ms at 0 mV. Recovery from inactivation at -80 mV was rapid, with a time constant of 252 ms. The A-type current was blocked by 4-aminopyridine (4-AP) and was inhibited by flecainide, with an IC(50) of 35 microM. The voltage for half-activation was -26 mV, while the voltage of half-inactivation was -65 mV. There was significant activation and incomplete inactivation at potentials positive to -60 mV, which is suggestive of sustained current availability in this voltage range. Under current-clamp conditions, exposure to 4-AP or flecainide depolarized the membrane potential by 7-10 mV. In intact antral tissue preparations, flecainide depolarized the membrane potential between slow waves by 5 mV; changes in slow waves were not evident. The effect of flecainide was not abolished by inhibiting enteric neurotransmission or by blocking delayed rectifier and ATP-sensitive K(+) currents. Transcripts encoding Kv4 channels were detected in isolated antral myocytes by RT-PCR. Immunocytochemistry revealed intense Kv4.2- and Kv4.3-like immunoreactivity in antral myocytes. These data suggest that the A-type current in murine antral smooth muscle cells is likely to be due to Kv4 channels. This current contributes to the maintenance of negative resting membrane potentials.

4-Aminopyridine↗